Edwin G. E. Jahngen

Creatine and Cyclocreatine Effects on Ischemic Myocardium: 31P Nuclear Magnetic Resonance Evaluation of Intact Heart

The purpose of this study was to investigate the effects of prior dietary supplementation with creatine (Cr) or cyclocreatine (Cy, a synthetic analogue of Cr) on high energy phosphate metabolism of the ischemic myocardium. To this end, 48 rats were fed the following powdered rat chow diet for 21 days: 16 were fed chow without additives (CON); 16 were fed a diet containing 1% Cr by weight (CR); 16 were fed a diet containing 1% Cy by weight (CY). At the end of the feeding period, rats were anesthetized, hearts harvested and perfused in the Langendorff mode using Krebs-Henseleit buffer (maintained at 37 degrees C, equilibrated with 95% O2/5% CO2) to which 11 mM glucose was added. 31P nuclear magnetic resonance (NMR) studies of myocardial bioenergetics were done using a Bruker AM 500 spectrometer. After acquisition of preischemic spectra, global ischemia was produced by clamping aortic inflow. Ischemia was maintained until adenosine triphosphate (ATP) became NMR invisible (CON = 34 +/- 11 min; CR = 32 +/- 13 min; CY = 56 +/- 13 min; p less than 0.05 CY vs. CR and CON). Half-lives of ATP were 19 min for CON and CR and 37.5 min for CY; half-lives of phosphagen were 4 min for CON and CR and 11 min for CY. Time for return of mechanical function (heart rate x systolic pressure) after ischemia was similar for all three groups (CON = 28 +/- 28, CR = 34 +/- 22, and CY = 22 +/- 15 min), even though the CY group was subjected to longer periods of ischemia). These data indicate that CY, but not CR, pretreatment provides myocardial protection either during and/or after ischemia and allows return of mechanical function after much longer episodes of ischemia than in CON and CR. One factor in the mechanism of protection may be the prolonged maintenance of phosphagen due to the higher equilibrium concentration of phosphocyclocreatine which in turn provides substrate for continued synthesis of ATP during and after ischemia, thus defining Cy as a bioenergetic protective agent. Other mechanisms of protection remain to be defined.

Intermediary purine-metabolizing enzymes from the cytosol of Dictyostelium discoideum monitored by high-performance liquid chromatography☆

The use of high-performance liquid chromatography to identify and quantitate five purine-metabolizing enzymes from a partially purified subcellular fraction of the eucaryotic microorganism Dictyostelium discoideum is described. All HPLC separations were carried out in an isocratic manner using reverse-phase C18 as the stationary phase. The mobile phase consisted of a phosphate buffer with either methanol or acetonitrile as cosolvent, and optimal separation conditions were attained by varying the organic concentration or the pH of the buffer or by employing paired-ion chromatographic techniques. Substrates and products were detected at either 254 nm for the purines or 295 nm for the formycin analogs. An adenosine kinase activity was identified, and it was demonstrated that formycin A (FoA) could be substituted for adenosine as the phosphate acceptor, yielding FoAMP as the product. With FoA as the substrate an apparent Km of 18.2 microM and an apparent Vmax of 32.4 mmol min-1 mg-1 were observed for the activity. A purine-nucleoside phosphorylase activity was found to cleave adenosine to adenine and ribosylphosphate. FoA was not found to be a substrate for this activity due to the unusual formycin C-glycosyl bond which was not hydrolyzed by enzymes or chemically with either HCl or NaOH. An adenylate deaminase activity was found to be present in the cytosolic S-100 of cells harvested during the onset of development, and this deaminase activity was greatly stimulated by ATP. With FoAMP as the substrate, an apparent Km of 236 microM and Vmax of 2.78 mumol min-1 mg-1 were observed. The deamination of FoAMP could be inhibited by the addition of the natural substrate AMP. An apparent Ki value of 136 microM was determined from initial rate data. An adenylosuccinate synthetase activity was observed to have a Km value for GTP, IMP, and aspartic acid of 23, 34, and 714 microM, respectively. The formycin analog FoIMP was not a substrate with this activity but was a competitive inhibitor of IMP. Finally hypoxanthine-guanine phosphoribosyltransferase was found to have Km and Vmax values for hypoxanthine of 55.5 microM and 34.3 nmol-1 min-1 mg-1. When guanine was used as the substrate, the rate of nucleotide formation was 50% that with hypoxanthine as the substrate. The advantages of using HPLC to examine the interconnecting activities of a multienzyme complex in subcellular fractions are discussed, including the increased sensitivity obtained by using formycin analogs in the assay procedures.(ABSTRACT TRUNCATED AT 400 WORDS)

LC-EC determination of nucleotides and nucleic acids: application to enzyme assays and the analysis of DNA fragments

High-performance liquid chromatography (HPLC) coupled to an electrochemical detector in an oxidative mode was used to analyze purine bases, nucleosides, and nucleotides as well as restriction fragments of nucleic acids. Ligands were separated by liquid chromatography with electrochemical detection (LC-EC) using size exclusion, ion-exchange, or reverse phase techniques. Using an amperometric electrochemical detector the determination was characterized with respect to sensitivity, selectivity, and capacity factor. It was observed from hydrodynamic and cyclic voltammetry that the optimum oxidation potential differed for the three major classes of purines, permitting an enhancement in selectivity when compared to detection. Guanylyl moieties demonstrated a half-wave potential at 0.800 V vs Ag/AgCl, while those for the adenylyl and inosylyl groups are above 1,000 V vs Ag/AgCl. The facility of the method to analyze components of a complex biological milieu was demonstrated by examining the purine pools of crude and partially purified eye lens homogenates as well as by comparing the traditional hexokinase assay to the newly developed LC-EC technique. Additionally, LC-EC was compared to detection for determination of the purine-metabolizing enzyme activities, adenylate deaminase and adenylosuccinate synthetase from crude cellular lysates of the cellular slime mold, Dictyostelium discoideum. Finally, the technique was used to assay the fragments from lambda-DNA cut with the restriction endonuclease Pst-1.

Environmentally benign synthesis of vinyl ester resin from biowaste glycerin

We present here for the first time a novel environmentally benign protocol for the synthesis of vinyl ester resin (VER). Our synthetic strategy utilizes a commercial waste material, glycerin, from biodiesel manufacturing and converts it into a widely utilized resin. The VER was synthesized using bisphenol A (BPA) and glycidyl methacrylate (GMA) as precursors. GMA was synthesized via a multistep synthetic protocol using glycerin obtained from a biodiesel manufacturing waste stream. The structure of the intermediates was confirmed by 1H NMR, HPLC and FT-IR spectroscopy.

ALPHA‐CARBALKOXYLATIONS OF CARBOXYLIC ACIDS, A GENERAL SYNTHETIC ROUTE TO MONOESTERS OF MALONIC ACIDS

Aus den Carbonsauren (I) entstehen mit Lithium-diisopropylamid die Li-Derivate (II), die mit Chlorkohlensaureester (III) oder Dialkylcarbonaten (V) zu den Malonester= Carbonsauren (IV) substituiert werden.

AMP deaminase in Dictycostelium discoideum: Increase in activity following nutrient deprivation induced by starvation or hadacidin

SummaryAMP deaminase, the activity that catalyzes the deamination of AMP to form IMP and NH3 has been measured in Dictyostelium discoideum. A new procedure to assay the activity of this enzyme was developed using formycin 5′-monophosphate, a fluorescent analog of AMP as the substrate, and ionpaired reverse phase HPLC to separate the reactants and products. Quantitation of the formycin containing compounds was accomplished at 290 nm. At this wavelength adenosine containing compounds were not detected and activity could be monitored in the presence of its activator ATP. The AMP deaminase activity in vegetative cells was 7.4 nmols/min/mg proteins while the activity in cells measured at 2 and 6 hrs after starvation-induced growth-arrest was 376 nmols/min/mg protein... a 51-fold increase. When vegetative cells were treated with hadacidin, a drug that restricts de novo AMP synthesis and pinocytosis, the activity of the AMP deaminase was 511 nmols/min/mg protein... a 70-fold increase compared to that in untreated vegetative cells. Smaller increases were noted following the inhibition of growth with the drugs cerulenin and vinblastine, as well as after the inhibition of de novo GMP synthesis with the drug mycophenolic acid or the partial inhibition of de novo AMP synthesis with analogs of hadacidin, N-hydroxyglycine and N-formylglycine. In addition, when the activity of two other enzymes involved in purine metabolism, namely adenosine kinase and hypoxanthine-guanine phosphoribosyl transferase, was measured in vegetative cells, and the activity of both compared to that measured in starvation and hadacidin induced growth-arrested cells, showed no significant changes. These data suggest that the changes in the activity of the AMP deaminase are in response to nutrient deprivation and further, that as a consequence of the increase in AMP deaminase activity, ammonia will be produced and an increase in pH should follow. The production of ammonia and its effect on development implicates the AMP deaminase in the early differentiation of this organism.

Terahertz absorption spectra of highly energetic chemicals

Research into absorption spectra is useful for detecting chemicals in the field. Each molecule absorbs a set of specific frequencies, which are dependent on the molecules structure. While theoretical models are available for predicting the absorption frequencies of a particular molecule, experimental measurements are a more reliable method of determining a molecules actual absorption behavior. The goal of this research is to explore chemical markers (absorption frequencies) that can be used to identify highly energetic molecules of interest to the remote sensing community. Particular attention was paid to the frequency ranges located within the terahertz transmission windows of the atmosphere. In addition, theoretical derivations, with the purpose of calculating the detection limits of such chemicals, will also be presented.

ALIGNED MULTIPLE-WALLED CARBON NANOTUBES AS BIOSENSORS BY COLORIMETRIC AND AMPEROMETRIC TECHNIQUES

Vertically oriented multiple-walled carbon nanotubes (MWCNTs) aligned on a silicon substrate were covalently bonded with horseradish peroxidase (HRP) and used as biosensors or electrodes. The modified platform was investigated with Scanning Electron Microscopy (SEM), UV-Vis spectrophotometer and Cyclic Voltammetry (CV). The enzyme catalyzed reaction and the subsequent reduction of the enzymatic products by the platform were carefully investigated. The assay was done by measuring the absorbance of the dye formed from the reaction product of the substrates (phenol) and 4-aminoantipyrine. From the linear response, phenol can be quantitized in the range from 47 ppm to 750 ppm with a detection limit of 19 ppm (based on S/N = 3). The resulting modified aligned MWCNTs still exhibited enzymatic activities after storage of 13 days. The activity of the attached HRP was also demonstrated electrochemically.

The effect of N-Nitrosonornicotine on the buccal mucosa of Syrian hamsters

PURPOSE The carcinogens in smokeless tobacco have been identified as the tobacco-specific nitrosamines and the effect of one of these, N-Nitrosonornicotine (NNN), on the buccal mucosa of the Syrian hamster was studied. MATERIALS AND METHODS Buccal pouches of 36 Syrian hamsters were painted five times per week for 24 weeks with 10 mg/mL 98% pure NNN in suspension with mineral oil. Animals were killed at 6, 8, 12, and 24 weeks. RESULTS At 6 weeks, the buccal pouch mucosa of the experimental animals appeared clinically more hyperemic than those of the controls. From 12 weeks onward, all experimental animals showed epithelial hyperplasia and inflammation on histologic examination. Three animals killed at 24 weeks showed mild epithelial dysplasia. CONCLUSIONS Exposure of Syrian hamster buccal mucosa to NNN, five times per week for 24 weeks, did not result in clinical or histologic cancerous changes. NNN may require other factors for cancer production, such as a cocarcinogen, a higher concentration, or a longer period of application.

Flow Stream Detectors Based on the Electrocatalytic Oxidation of Polyhydroxy Compounds at Silver Oxide Electrodes

Many simple carbohydrates and other polyhydroxy compounds can be oxidized at a silver oxide surface. The oxidation is via an electrocatalytic mechanism involving a Ag(I) oxide. This forms the basis of a flow stream detector operated in an amperometric mode which may be used for either flow injection or high performance liquid chromatography (HPLC) applications. The title electrode has been applied to the detection of simple carbohydrates, triglycerides and nucleic acid components.